PROJECT SUMMARY CRISPR-Cas12a has recently emerged as a powerful gene editing tool with great potential to ameliorate wide- ranging diseases through gene therapy. Cas12a, like Cas9, is a nuclease that can be programmed to cut genomes at specific sequences with high efficiency, but it is better for targeting AT-rich sequences and multiple genes simultaneously. The safe implementation of Cas12a, however, requires the development of inhibitors that can enable regulation and prevent editing at off-target sites. It also requires improved understanding of Cas12a biology and cleavage activity in cells, for which a paucity of data exists. For example, Cas12a has been shown to indiscriminately cleave single-stranded DNA (i.e. perform trans-cleavage) after binding to its target DNA in vitro, but it is not known if this occurs in cells. The long-term objectives of this proposal are to identify and develop Cas12a inhibitors and determine if Cas12 trans-cleavage occurs in vivo. Using bioinformatics and in vivo assays, we have recently discovered the first three proteins (acrVA1-3) that inhibit Cas12a cleavage in bacteria and in human cells. These proteins are encoded in a phage (virus) infecting bacteria, where they inhibit phage cleavage by Cas12a. These inhibitors stand to provide useful tools for Cas12a regulation, but their successful implementation requires insight into their mechanisms of inhibition. Preliminary evidence suggests that each AcrVA protein functions by a distinct mechanism, which will be elucidated using a variety of in vitro and in vivo assays that determine their effect on Cas12a expression and target DNA binding. Next, we will identify AcrVA proteins that optimally inhibit different Cas12a variants commonly used in gene editing. This will be achieved by mutagenizing acrVA1 and selecting for optimized inhibitors using bacterial selection screens as well as by exploring natural acrVA diversity using bioinformatics and in vivo inhibition assays. Finally, the existence of indiscriminate Cas12a trans-cleavage in vivo and its susceptibility to inhibition by acrVA1-3 will be determined using phage infection experiments in bacteria. Overall, this work will illuminate fundamental Cas12a biology and develop these novel inhibitors into powerful tools that can regulate Cas12a activity. In doing so, it will significantly improve the safety and utility of Cas12a in correcting genetic disorders. This work will be performed at UCSF, which hosts world-class facilities and a highly intellectual and collaborative research community. It will also provide me with the expertise in bacterial-phage biology, biochemistry, and gene editing that I need to fulfill my postdoctoral training goals and pioneer an independent research program in bacterial-phage counter-immunity.